摘要
目的:探讨活性氧(ROS)对人类精子线粒体tRNALeuUUR基因的氧化损伤。方法:采用Percoll梯度离心法筛选具有正常生理功能的精子,作为正常精子模型,并分为损伤组20例和对照组20例,分别加入次黄嘌呤-黄嘌呤氧化酶体系或不予处理,37℃有氧环境中孵育60min。分别提取精子DNA,以Fpg酶切损伤碱基并采用接头介导PCR(LM-PCR)检测线粒体tRNALeuUUR基因的氧化损伤。采用Rhodamine(Rh123)荧光探针标记精子,通过流式细胞仪检测线粒体膜电位,观察精子的功能。结果:与对照组相比,损伤组精子孵育后线粒体膜电位明显降低[(116.27±11.72)%vs(64.00±4.88)%,P<0.05]。Fpg酶切和LM-PCR显示精子线粒体tRNALeuUUR基因损伤。结论:ROS可能通过对精子线粒体tRNALeuUUR基因氧化损伤而影响精子功能(线粒体膜电位明显降低),从而引起不育。
Objective： To study the oxidative damage to human sperm mitochondrial tRNA^LeuUUR gene by reactive oxygen species （ROS） in vitro. Methods： Spermatozoa of normal physiological function selected from semen samples by Percoll gradient centrifugation technique were used as normal sperm models, which were divided into two groups of 20 cases eaeh, a damage group and a control group, the former treated with hypoxanthine xanthine oxidase system and the latter left untreated, both incubated at 37℃ in aerobic environment for 60 minutes. Sperm DAN was extracted, and digestion by the enzymes fpg and ligation-mediated PCR （LM-PCR） was performed to map the damage to mitochondrial tRNALeuUUR gene. The spermatozoa were labeled with specific fluorescent probe of Rhodamine 123 to measure mitochondrial membrane potential （MMP） by flow cytometry and observe sperm function. Results： Compared with the control group, after the normal spermatozoa were incubated with ROS, MMP of the spermatozoa significantly decreased （ 116.27 ± 1.72 vs 64.00±4.88 ） , P 〈 0.05. Digestion by the enzymes fpg and LM-PCR showed damage to mitochondrial tRNA^LeuUUR gene. Conclusion： Reactive oxygen species may inflict oxdative damage on sperm mitochondrial tRNA^LeuUUR gene and thus affect sperm function（ as shown by significant decrease in MMP） , resulting in infertility.
出处
《中华男科学杂志》
CAS
CSCD
2006年第12期1084-1087,共4页
National Journal of Andrology
